Dipolar compounds, that is to say, molecules that exhibit both one or more positive and negative formal charges within the same structure, are known as zwitterions. At a pH specific to a particular zwitterion, known as the isoelectric point (pI), the net charge of that zwitterion is zero. In aqueous solution, zwitterions are typically least soluble at the isoelectric point. In biological systems, α-amino acids are among the most common zwitterionic molecules. The simplest amino acid is glycine, which has no side groups. In acidic solutions, both the α-amino group and the carboxylic acid group of glycine are protonated. At the isoelectric point, the α-amino group is protonated, but the carboxyl group is ionized. In basic solutions, both the α-amino group and the carboxyl group are deprotonated. Some natural amino acids, such as lysine, arginine and histidine, have basic side chains, and thus undergo multiple ionization states. Most naturally occurring amino acids may be precipitated by titration with appropriate acidic or basic solutions, such as hydrochloric acid or aqueous sodium hydroxide, respectively, until the isoelectric point, and hence the lowest solubility are achieved. In addition, removal of excess cationic counterion from such a titration may be performed by adding a sufficient amount of strong base. For example, to remove hydrochloride cations from an amino acid, an amount of sodium hydroxide may be added to form the sodium salt of the amino acid.
Nitric oxide (NO) is a bioactive free radical gas produced by any one of several isoforms of the enzyme nitric oxide synthase (NOS). The physiological activity of what was later identified as NO was initially discovered in the early 1980's when it was found that vascular relaxation caused by acetylcholine is dependent on the presence of the vascular endothelium. The factor derived from the endothelium, then called endothelium-derived relaxing factor (EDRF), that mediates such vascular relaxation is now known to be NO that is generated in the vascular endothelium by one isoform of NOS. The activity of NO as a vasodilator has been known for well over 100 years. In addition, NO is the active species derived from known nitrovasodilators including amylnitrite, and glyceryltrinitrate. Nitric oxide is also an endogenous stimulator of soluble guanylate cyclase (cGMP), and thus stimulates cGMP production. When NOS is inhibited by N-monomethylarginine (L-NMMA), cGMP formation is completely prevented. In addition to endothelium-dependent relaxation, NO is known to be involved in a number of biological actions including cytotoxicity of phagocytic cells and cell-to-cell communication in the central nervous system.
The identification of EDRF as NO coincided with the discovery of a biochemical pathway by which NO is synthesized from the amino acid L-arginine by the enzyme NO synthase. There are at least three types of NO synthase as follows:                (i) a constitutive, Ca++/calmodulin dependent enzyme, located in the brain, that releases NO in response to receptor or physical stimulation;        (ii) a Ca++ independent enzyme, a 130 kD protein, which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a number of other cells by endotoxin and cytokines; and        (iii) a constitutive, Ca++/calmodulin dependent enzyme, located in the endothelium, that releases NO in response to receptor or physical stimulation.        
Once expressed, inducible nitric oxide synthase (hereinafter “iNOS”) generates NO continuously for long periods. Clinical studies have shown that NO production and iNOS expression are increased in a variety of chronic inflammatory diseases, such as rheumatoid and osteoarthritis (see, e.g, McInnes I. B. et al., J. Exp. Med. 184:1519 (1996)), inflammatory bowel disease (see, e.g, Lundberg J. O. N. et al., Lancet 344:1673, (1994)), and asthma (see, e.g., Hamid, Q. et al., Lancet 342:1510 (1993)), and iNOS is implicated as a major pathological factor in these chronic inflammatory diseases.
Thus, inhibition of excessive NO production by INOS is likely to be anti-inflammatory. However, since the production of NO from eNOS and nNOS is involved in normal physiology, it would be desirable for any NOS inhibitor that is used for treating inflammation be selective for iNOS, so that normal physiological modulation of blood pressure by eNOS-generated NO, and non-adrenergic, non-cholinergic neuronal transmission by nNOS-generated NO would remain unaffected.
With all pharmaceutical compounds and compositions, the chemical and physical stability of a drug compound is important in the commercial development of that drug substance. Such stability includes the stability at ambient conditions, especially to moisture and under storage conditions. Elevated stability at different conditions of storage is needed to predict the different possible storage conditions during the lifetime of a commercial product. A stable drug avoids the use of special storage conditions as well as frequent inventory replacement. A drug compound must also be stable during the manufacturing process which often requires milling of the drug to achieve drug material with uniform particle size and surface area. Unstable materials often undergo polymorphic changes. Therefore, any modification of a drug substance which enhances its stability profile provides a meaningful benefit over less stable substances.
Several inhibitors of iNOS have been described, such as, for example, S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, which is described and claimed in commonly assigned U.S. Pat. No. 6,403,830. That compound, however, is an amorphous solid. It would be desirable, therefore, to provide a crystalline solid form of an iNOS inhibitor such as S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine.
Other iNOS inhibitors compounds that are synthetic amino acid analogs that include amidine functional groups include: S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochloride; S-[2-(ethanimidoylamino)-1-methylethyl]cysteine; (2S,5E)-2-amino-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoic acid, dihydrochloride; (S, E)-2-amino-2-methyl-6-[(1-iminoethyl)amino]-4-hexenoic acid, dihydrochloride; (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid, dihydrochloride; and (2S,5E)-2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoic acid, dihydrochloride.
Unfortunately, traditional methods of removal of cationic counterions by titration with a strong base generally results in decomposition of the amidine functional group of these compounds.